Progesterone is the major secretory product of the corpus luteum (CL). Deficiency in either the duration or the amount of progestin secretion leads to an impairment of the endometrial development and its ability to support pregnancy. Progesterone and its synthetic analogues (progestins) are being widely used to restore the normalcy of the endometrium in a variety of clinical conditions including CL defects, habitual and threatened abortions, endometrial hyperplasias, and certain forms of endometrial cancers [Chez, R. A. Fertil. Steril., Vol. 30, p. 16 (1978)]. They are also employed as contraceptive agents in a variety of delivery systems [Landgren, B. M., Lager, S., and Diczfalusy, E., Contraception, Vol. 23, p. 269 (1981); Somboonsuk, A., Israngkum, C., Siriwongse, T., Dusitin, N., Onthuam, Y., Chaudhary, R. R., Fuchs, F., Grossman, R., and Gray, R., Contraception, Vol. 18, p. 137 (1978); Newton, J., Szontagh, F., Lebech, P., and Rowe, P., Contraception, Vol. 19, p. 575 (1979); Toivonen, J., Contraception, Vol. 20, p. 511 (1979); Kurunmaki, H., Toivonen, J., Lahteenmaki, P. and Luukkainen, T., Contraception, Vol. 23, p. 473 (1981)]. In spite of such widespread use of progestins, very little is known about their biochemical effects on the human endometrium and the mechanisms by which these effects are produced. Moreover, the art lacks reliable means of monitoring the responses of endometrium in individual patients to endogenous or exogenous progestins.
Progesterone has been shown to bring about the differentiation of estrogen-dominated proliferative phase tissue into a secretory phase tissue. Continued stimulation of this target tissue with progesterone, as occurring in early pregnancy, promotes decidualization of the endometrial stromal cells and induces a hyper-secretory response within the glands. Studies on the mechanism of progestin action which have been conducted on animal models have clearly shown that progesterone, like many other steroid hormones, induces specific changes in the transcription and translation of the genetic messages encoded in the cellular DNA of the target cells [Glasser, S. R., Clark, J. H., Smith, R. G. and O'Malley, B. W. "Mechanism of Steroid Hormone Action in `Endocrinology of Pregnancy`". Eds. F. Fuchs and Klopper, A. Harper and Row, New York (1977), pp. 15-40; Beier, H. M., Biochim. Biophys. Acta., Vol. 160, p. 289 (1968); Squire, G. D., Bazer, F. W. and Murray, F. A., Biol. Reprod., Vol. 7, p. 321 (1972); Laster. D. B., Biol. Reprod., Vol. 16, p. 682 (1977)]. Specific progestin-dependent translation products such as uteroglobin, purple protein and an acidic protein, and a pregnancy-specific protein have been detected in the reproductive tract of laboratory animals by other investigators. However, such proteins are species-specific, and hence, of limited clinical utility.
Based on earlier evidence, we postulated that the action of progestin on the endometrium involves the preferential synthesis of a protein(s) which plays a key role in pregnancy. Analysis of the protein patterns of the progestin-dominated human endometrium led us to the detection of such a specific protein which we have designated "progestagen-associated endometrial protein", or PEP. The detection of such a PEP was first reported by us in May 1976 at the New York Academy of Science-sponsored Conference on Biochemical Actions of Progesterone and Progestins [Joshi, S. G. in Discussion (pp. 246-247) of the paper entitled "Protein Composition of Human Endometrium and its Secretion at Different Stages of Menstrual Cycle", by Hirsh, P. M., Fergusson, I. L. and King R. J. B., Ann. N.Y. Acad. Sci. Vol. 286, p. 233, (1977)]. A more detailed account of PEP was presented at the 10th Annual Meeting of the Society for the Study of Reproduction, held in 1977 in Austin, Tex., U.S.A. [Joshi, S. G., Ebert, K. M., and Swartz, D. P., Program of the 10th Annual Meeting for the Society for the Study of Reproduction, 1977. (Abstract No. 124)].
Our studies demonstrate that the endometrium is the major site of PEP synthesis and that PEP is formed within the endometrial glands and secreted into aminotic fluid and blood. We found that, in non-pregnant women, serum PEP levels reflect the stage of endometrial development, rather than the ovarian activity. In infertile women, induction of CL formation, but not stimulation of ovarian follicular activity, is associated with high serum PEP levels. IN normally cycling women, serum PEP levels increase markedly after ovulation reaching a peak during the late secretory phase. Synthesis of PEP is increased dramatically during early pregnancy (8-10 weeks) as indicated by more than 1000-fold and 10-fold increases in PEP levels in endometrial tissue and blood, respectively. Serum PEP level declines rapidly after about 14 weeks of pregnancy indicating the onset of degeneration of the endometrial glands.
Although the precise role of PEP in pregnancy has not been finally determined, the involvement of PEP in biochemical reactions that occur during implantation, invasion of the endometrium by trophoblasts and during immuno-suppression is being studied. Finally and, most importantly, our studies demonstrate that serum PEP represents a steroid-specific biochemical marker of the cumulative effect of progestins on the human endometrium. It is apparent, therefore, that serum PEP determination will prove to be a very valuable tool in the clinical practice: (i) to diagnose CL defects; (ii) to monitor the response of endometrium to endogenous or exogenous progestins in normal or abnormal pregnancies; and (iii) to predict response of endometrial hyperplasias or neoplasias to progestin therapy.
During our investigation, we developed several immunological methods for the detection of PEP. These included Ouchterlony's double diffusion test [Joshi, S. G., Ebert, K. M., and Swartz, D. P., J. Reprod. Fert., Vol. 59, p. 273 (1980)], Scheidegger's micro immunoelectrophoresis [Joshi et al, supra], radioimmunoelectrophoresis [Joshi, S. G., Bank, J. F., and Szarowski, D. H., J. Clin. Endocr. Metab., Vol. 52, p. 1185 (1981)], polyacrylamide gel electrophoresis combined with immunodiffusion [Joshi, S. G., Ebert, K. M., and Swartz, D. P., J. Reprod. Fert., Vol. 59, p. 273 (1980)], and immunoperoxidase staining [Mazurkiewicz, J. E., Bank, J. F., and Joshi, S. G., J. Clin. Endocr. Metab., Vol. 52, p. 1006 (1981)]. All of these tests utilized rabbit and goat antisera which were generated against partially purified proteins of the decidua (DE) of early pregnancy. Before use, such anti-PEP sera were thoroughly absorbed with human adult male serum and/or with cytosol of term placenta to remove contaminating antibodies [Joshi, S. G., Ebert, K. M., and Swartz, D. P., J. Reprod. Fert., Vol. 59, p. 273 (1980); Joshi, S. G., Bank, J. F., and Szarowski, D. H., J. Clin. Endocr. Metab., Vol, 52, p. 1185 (1981); Mazurkiewicz, J. E., Bank, J. F., and Joshi, S. G., J. Clin. Endocr. Metab., Vol. 52, p. 1006 (1981)]. A preliminary screening of PEP in endometrial tissues in different developmental stages by immunodiffusion test and immunoelectrophoresis demonstrated the presence of PEP in all of forty-two specimens of DE (unavoidably contaminated with trophoblasts, fetal tissues and blood), in all of eight specimens of uncontaminated DE, in one decidua of tubal pregnancy, and in eighteen of thirty-eight samples of the proliferative phase endometria (PE) of cycling women, or in pregnancy or non-pregnancy sera [Joshi, S. G., Ebert, K. M., and Swartz, D. P. Program of the 10th Annual Meeting for the Society for the Study of Reproduction, 1977. (Abstract No. 124)].
A study [Joshi, S. G., Ebert, K. M., and Smith, R. A., J. Reprod. Fert., Vol. 59, p. 287 (1980)] revealed that PEP is heterogeneous in several respects; in addition to being precipitated over a wide range of ammonium sulfate concentrations, it splits into different populations during chromatography on DEAE-cellulose or on hydroxypatite. Furthermore, during polyacrylamide gel electrophoresis and isoelectric focusing on acrylamide gels, PEP separates as a diffuse rather than a sharp band. PEP is a negatively charged protein with alpha mobility, and on acrylamide gels, it migrates in the post-albumin region. PEP is a glycoprotein which binds to Concanavalin-A. The immunologic reactivity of PEP is retained after 30-minute exposure to 4.degree.-85.degree. C. at pH 7.4 or after 2 hours to pH 2 to 11 at 22.degree. C. Trypsin, but not pepsin, RNase, DNase or neuraminidase, completely destroys its immunoreactivity. The apparent molecular weight (MW), as determined by filtration on Sephadex G-100 or Sephacryl S-200 (Pharmacia), was found to be 47,000. PEP can be reductively dissociated into two subunits, each having MW of approximately 27,000. The isoelectric point determined by focusing on polyacrylamide gels was found to be approximately 4.9 Analysis of the cell fractions of DE homogenates obtained by the differential centrifugation method has shown the presence of PEP in the crude nuclei, heavy particulate fraction (15,000 xg, 20 minutes, sediment) and in cytosol but not in the light particulate fraction (130,000 xg, 90 minutes, sediment). Immunoperoxidase staining was employed to localize PEP in endometrial tissue, and it was found to be localized exclusively in glandular epithelial cells and within the glandular lumen.
An investigation was conducted to determine whether PEP is indeed synthesized within the endometrium, and if so, whether the rate of PEP synthesis is altered during the progestational phase development of the endometrium. This was determined by the conventional in vitro double-labeling technique. Briefly the technique involved labeling proteins of the endometrium in one developmental stage (e.g., proliferative phase) with .sup.14 C-leucine and proteins of the endometrium in another developmental stage (e.g., secretory or decidual phase) with .sup.3 H-leucine. After labeling, aliquots of soluble proteins derived from the endometria in the two developmental stages were combined and the mixtures (containing .sup.14 C- and .sup.3 H-labeled proteins) were treated with anti-PEP serum to precipitate PEP or they were electrophoresed on polyacrylamide gels in absence or presence of sodium dodecyl sulfate (native or SDS-gels). The results of the immunoprecipitation experiments showed that the ratios of .sup.3 H/.sup.14 C (Exps. 1-4) or .sup.14 C/.sup.3 H in the immunoprecipitates containing PEP were markedly higher than those in the original mixtures. The electrophoretic studies demonstrated that the ratios of .sup.3 H/.sup.14 C in those segments of the acrylamide gels containing PEP were markedly higher than those in other segments of gels that did not contain PEP. It was concluded, therefore, that the rate of PEP synthesis is markedly higher in the secretory and in the decidual phase endometrial than in the proliferative phase endometria. In addition, electrophoretic studies on radio-proliferative endometrium also synthesized PEP but in amounts below the detection limit of immunodiffusion or immunoelectrophoresis.
It also became important to know whether the endometrium is the only or the major source of PEP in human females. Therefore studies were undertaken to study distribution of PEP in tissues and body fluids.
PEP could not be detected by immunoelectrophoresis or immunodiffusion in the pregnancy or non-pregnancy sera, the extracts of early-, mid- or full-term placentae, umbilical cord, and the extracts of ovaries, Fallopian tubes, myometria and cervixes that were dissected from the total abdominal hysterectomy specimens of women in proliferative secretory phase of menstrual cycle. Also PEP could not be detected in the extracts of kidney, liver and spleen or young women. However, PEP was readily detected by the relatively insensitive immunodiffusion technique in the secretory- and decidual-phase endometria of cycling and pregnant women and in the amniotic fluid samples obtained during 15-18 weeks of pregnancy [Joshi, S. G., Smith, R. A. and Stokes, D. K., J. Reprod. Fert., Vol. 60, p. 317 (1980)]. We came to the realization that definite conclusions regarding the tissue-specificity of PEP could not be drawn unless more sensitive methods, such as a radioimmunoassay or an immunocytochemical technique, were developed for its detection in tissues or in specific cell types. Nevertheless, the studies conducted strongly suggest that the endometrium is the major, if not the sole, source of PEP in women.
Highly sensitive radioimmunologic tests demonstrate that PEP is not related to any of the following plasma, placental or uterine proteins: transferrin, .alpha.-1-anti-trypsin, ceruloplasmin, human prolactin, human chorionic gonadotropin, human placental lactogen, placental protein SP.sub.1, pregnancy zone protein, human .alpha.-fetoprotein, pregancy-associated plasma protein (PAPP's) and uteroglobin. However, we have recently found that PEP is serologically identical to alpha-uterine protein (AUP) which was independently detected in amniotic fluid by Sutcliffe in 1976 [Sutcliffe, R. G., Joshi, S. G., Paterson, W. F. and Bank, J. F., communicated to J. Reprod. Fert. In Press; Sutcliffe, R. G., Proteides Biol. Fluids, Vol. 24, p. 543 (1976)]. However, the progestin-dependency of AUP was not studied by Sutcliffe.
The clinical applications of PEP depend heavily on the ability to detect and quantitate PEP in biological fluids and tissues which can be readily retrieved by relatively non-invasive means.
Heretofore there existed only one method for the quantitation of PEP in human tissues and body fluids, i.e., rocket immunoelectrophoresis (RIEP) (Joshi, S. G., Henriques, E. S., Smith, R. A., and Szarowski, D. H., Am. J. Obstet. Gynecol., 138, p. 1131 (1980), Sutcliffe, R. G., Brock, D. J. H., Nicholson, L. V. B. and Dunn, E. J., Reprod. Fertil. 54, p. 85 (1978).
It is an object of the present invention to provide radioimmunoassay methods for the detection of PEP in body fluids which are far more sensitive and vastly superior to the methods employed heretofore for the detection thereof.
It is a further object of the present invention to provide unlabeled PEP and a radioisotope labeled PEP useful in the above-noted radioimmunoassay methods.
It is a further object of the present invention to provide a method for the preparation of an antibody against PEP and a method for testing the quality thereof, said antibody also being useful in the above-noted radioimmunoassay method.
It is a further object of the present invention to provide a composition in kit form especially adapted for conducting the above-noted radioimmunoassay method.
It is a further object of the present invention to provide methods for diagnosing conditions of the human body which affect the production or maintenance of response of the endometrium to endogenous or exogenous progestins.